The long term objective of this proposal is to understand the molecular basis of the abnormal energy metabolism of many transformed cells, which is characterized phenotypically by a high glucose catabolic rate. The major focus will be on the enzyme hexokinase which catalyzes the first and committed step of glucose catabolism. Significantly, hexokinase is markedly elevated in many transformed cells where it targets mitochondrial receptors. Recent progress has shown that hexokinase is the only glycolytic enzyme in tumor cells bound to the mitochondria, where it serves to preferentially phosphorylate glucose with mitochondrially generated ATP (Can. Res., 1988; JBC, 1988). The purification of the tumor enzyme and its mitochondrial receptor have been completed and a full-length cDNA of the enzyme has been cloned, sequenced and overexpressed in active form in E. coli (Biochem., 1986; Can. Res., 1988; JBC, 1990a, 1990b). Some very fundamental questions can now be addressed about the tumor hexokinase molecule, its capacity for mitochondrial binding, and its high expression during tumor cell growth. Specific Aims are six-fold: 1. To clearly define, using deletion analysis and synthetic peptides, those regions of the tumor hexokinase molecule involved in mitochondrial binding, catalysis, and regulation by Glu-6-P. 2. To identify, using random and site directed mutational analysis, those amino acids most critical for binding the tumor hexokinase molecule to the outer mitochondrial membrane. 3. To establish, using approaches outlined in "1" and "2" above, the relationship between that region of the tumor hexokinase molecule involved in membrane binding and that region involved in ATP-dependent enzyme release. 4. To elucidate the molecular basis of the regulatory process in transformed cells that is primarily responsible for the high "net" expression of the gene coding for mitochondrial hexokinase. 5. To determine to what extent tumor promoters and growth regulators induce normal cells to take on the cancer phenotype in which hexokinase levels, mitochondrial binding, and high glucose catabolic rates correlate. 6. To elucidate the mechanism of the antitumor drug Lonidamine which inhibits mitochondrial bound hexokinase and glucose catabolism in certain tumor cell lines. The proposed research is not only of fundamental significance to our understanding of glucose catabolism and its regulation, in normal and transformed cells, but may provide new insights into the design of antitumor drugs that target energy metabolism.